Dysregulation of microRNA-204 mediates migration and invasion of endometrial cancer by regulating FOXC1.

MicroRNAs (miRNAs) regulate mRNA stability and protein expression, and certain miRNAs have been demonstrated to act either as oncogenes or tumor suppressors. Differential miRNA expression signatures have been documented in many human cancers but the role of miRNAs in endometrioid endometrial cancer (EEC) remains poorly understood. This study identifies significantly dysregulated miRNAs of EEC cells, and characterizes their impact on the malignant phenotype. We studied the expression of 365 human miRNAs using Taqman low density arrays in EECs and normal endometriums. Candidate differentially expressed miRNAs were validated by quantitative real-time PCR. Expression of highly dysregulated miRNAs was examined in vitro through the effect of anti-/pre-miRNA transfection on the malignant phenotype. We identified 16 significantly dysregulated miRNAs in EEC and 7 of these are novel findings with respect to EEC. Antagonizing the function of miR-7, miR-194 and miR-449b, or overexpressing miR-204, repressed migration, invasion and extracellular matrix-adhesion in HEC1A endometrial cancer cells. FOXC1 was determined as a target gene of miR-204, and two binding sites in the 3'-untranslated region were validated by dual luciferase reporter assay. FOXC1 expression was inversely related to miR-204 expression in EEC. Functional analysis revealed the involvement of FOXC1 in migration and invasion of HEC1A cells. Our results present dysfunctional miRNAs in endometrial cancer and identify a crucial role for miR-204-FOXC1 interaction in endometrial cancer progression. This miRNA signature offers a potential biomarker for predicting EEC outcomes, and targeting of these cancer progression- and metastasis-related miRNAs offers a novel potential therapeutic strategy for the disease.

CHD5 Downregulation Associated with Poor Prognosis in Epithelial Ovarian Cancer.

BACKGROUND: The CHD5 gene located on 1p36 encodes a protein-chromodomain helicase DNA-binding protein 5. CHD5 has been shown to be a tumor suppressor gene candidate. This study investigated the involvement of CHD5 in ovarian cancer and its clinicopathological significance. METHODS: CHD5 expression in ovarian cancer and its counterpart were determined by quantitative RT-PCR. The correlation of CHD5 expression to clinicopathological features of the tumor was analyzed. RESULTS: CHD5 expression was downregulated by at least twofold in 32 of 72 (41%) invasive epithelial ovarian carcinomas when compared to 12 controls in Hong Kong Chinese women. CHD5 downregulation was correlated to clinical status (p < 0.05), but not to patient age, tumor type and grade, recurrence and clinical stage (p > 0.05). Survival analysis showed that patients with CHD5 downregulation in their tumors were associated with shorter disease-free and total survival times compared to those without CHD5 downregulation (p < 0.05). Cox proportional-hazards regression analysis indicated that downregulation of CHD5 is an independent adverse prognostic factor in ovarian cancer. CONCLUSION: This study shows that CHD5 is downregulated in a certain number of ovarian cancers and appears to be an adverse predictor candidate of ovarian cancer disease-free and total survival.

Identification of molecular markers and signaling pathway in endometrial cancer in Hong Kong Chinese women by genome-wide gene expression profiling.

Endometrial cancer is the third most common gynecologic malignancy and the ninth most common malignancy for females overall in Hong Kong. Approximately 80% or more of these cancers are endometrioid endometrial adenocarcinomas. The aim of this study was to reveal genes contributing to the development of endometrioid endometrial cancer, which may impact diagnosis, prognosis and treatment of the disease. Whole-genome gene expression analysis was completed for a set of 55 microdissected sporadic endometrioid endometrial adenocarcinomas and 29 microdissected normal endometrium specimens using the Affymetrix Human U133 Plus 2.0 oligonucleotide microarray. Selected genes of interest were validated by quantitative real-time-polymerase chain reaction (qRT-PCR). Pathway analysis was performed to reveal gene interactions involved in endometrial tumorigenesis. Unsupervised hierarchical clustering displayed a distinct separation between the endometrioid adenocarcinomas and normal endometrium samples. Supervised analysis identified 117 highly differentially regulated genes (>or=4.0-fold change), which distinguished the endometrial cancer specimens from normal endometrium. Twelve novel genes including DKK4, ZIC1, KIF1A, SAA2, LOC16378, ALPP2, CCL20, CXCL5, BST2, OLFM1, KLRC1 and MBC45780 were deregulated in the endometrial cancer, and further validated in an independent set of 56 cancer and 29 normal samples using qRT-PCR. In addition, 10 genes were differentially regulated in late-stage cancer, as compared to early-stage disease, and may be involved in tumor progression. Pathway analysis of the expression data from this tumor revealed an interconnected network consisting of 21 aberrantly regulated genes involved in angiogenesis, cell proliferation and chromosomal instability. The results of this study highlight the molecular features of endometrioid endometrial cancer and provide insight into the events underlying the development and progression of endometrioid endometrial cancer.

The comparative study of Sprague-Dawley and Lewis rats in adjuvant-induced arthritis.

The outbred Sprague-Dawley (SD) rats, similar to the inbred Lewis (LEW) rats, have been recently demonstrated to be highly susceptible to adjuvant-induced arthritis (AIA). We herein compared AIA in SD and LEW rats in terms of clinical, histological, radiological, and immuno-inflammatory features. The results showed that, following inoculation with a ground Mycobacterium tuberculosis (MT) suspension, SD and LEW rats manifested closely similar disease progression, with 100% incidence and similar severity. The development of arthritis was accompanied by significantly higher erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels than in control rats. Radiographic examination of the hind paws showed that both SD and LEW AIA rats manifested conspicuous soft tissue swelling, bone matrix resorption, periosteal new bone formation and bone erosion, while histopathological analysis of the synovial joints revealed marked cellular infiltration, angiogenesis, synovial hyperplasia, pannus formation, narrowing of joint space, and cartilage and bone destruction. Moreover, in relation to disease progression, serum tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 levels were markedly overexpressed in both SD and LEW AIA versus control rats, and SD and LEW AIA rats exhibited divergent profiles for the expression of TNF-alpha and IL-1beta. Taken together, these results demonstrated that the SD rat AIA model shares several arthritic features with the comparable model in LEW rats. Hence, given the more favorable characteristics of SD rats than LEW rats (i.e., lower cost, wider availability, and heterogenic background), this SD rat AIA model is more cost effective and advantageous for screening and testing novel anti-arthritic agents.

Integrations of the hepatitis B virus (HBV) and human papillomavirus (HPV) into the human telomerase reverse transcriptase (hTERT) gene in liver and cervical cancers.

Chronic infections with the hepatitis B virus (HBV) and high-risk human papillomaviruses (HPVs) are important risk factors for hepatocellular carcinoma (HCC) and cervical cancer (CC), respectively. HBV and HPV are DNA viruses that almost invariably integrate into the host genome in invasive tumors. The viral integration sites occur throughout the genome, leading to the presumption that there are no preferred sites of integration. A number of viral integrations have been shown to occur within the vicinity of important cancer-related genes. In studies of HBV-induced HCC and HPV-induced CC, we have identified two HBV and three HPV integrations into the human telomerase reverse transcriptase (hTERT) gene. Detailed characterization of the integrations revealed that four integrations occurred within the hTERT promoter and upstream region and the fifth integration occurred in intron 3 of the hTERT gene. None of the integrations altered the hTERT coding sequence and all resulted in juxtaposition of viral enhancers near hTERT, with potential activation of hTERT expression. Our work supports the hypothesis that the sites of oncogenic viral integration are nonrandom and that genes at the sites of viral integration may play important roles in carcinogenesis.

Frequent loss of heterozygosity of chromosome 3 short arm detected by PCR-based microsatellite polymorphisms in cervical squamous cell carcinoma.

Karyotypic studies have shown that genetic aberrations of the short arm of chromosome 3 (3p) may be involved in the pathogenesis of cervical carcinoma. In this study we analyzed nine polymorphic microsatellite repeats on 3p using a PCR-based assay for loss of heterozygosity (LOH) in 64 invasive squamous cell carcinomas of the cervix. These markers encompass chromosome region 3p13-25. LOH at one or more loci was detected in 46 (79%) out of the 58 informative cases. The incidence of LOH at locus D3S643 (3p13) was the highest among nine markers examined. The difference between the frequency of LOH at D3S643 in early stage (I-II) disease (43%) and those with advanced stage (stage III-IV) (79%) was statistically significant (P < 0.05). The results indicate that tumor suppressor gene(s) that play a role in cervical cancer may be located on the short arm of chromosome 3, likely near or at 3p13. The LOH at 3p13 appears to be a late event in tumor progression and may serve as an indicator for a less favorable clinical outcome.

Expression of apoptotic regulators and their significance in cervical cancer.

Insufficient apoptosis is implicated in many human cancers, including cervical carcinoma. The objectives of this study were to explore changes of apoptosis-regulating gene expression and their clinical significance in cervical cancer. The expression of apoptosis-regulating genes, including five Bcl-2 family and two caspase family members, was evaluated in 43 cervical invasive squamous cell carcinomas, using immunohistochemistry. Specimens in which >or=10% of the neoplastic cells showed cytosolic immunoreactivity were considered to be immunopositive. Results were correlated with clinico-pathologic characteristics of the subjects. All seven apoptotic regulators examined were positive in a proportion of the tumors. The percentage of cases expressing Bax was higher in the patients without evidence of disease after treatment than in the patients alive with disease or who died of disease (P<0.05). A significant difference in disease-free survival was detected between Bax-positive and -negative groups (P<0.05), and in overall survival between Mcl-1-positive and -negative groups (P<0.05). Significant association between the seven markers tested was only found for caspase 3 and Bak immunoreactivity in cervical carcinoma (P<0.05). The results demonstrate expression of multiple apoptosis-modulating proteins in cervical cancer. There appears to be complex regulation of apoptosis protein levels in association with clinical behavior of cervical squamous cell carcinoma.

Frequent ras gene mutations in squamous cell cervical cancer.

Eighty samples of cervical invasive squamous cell carcinoma were examined for ras gene mutations using polymerase chain reaction (PCR) followed by restriction enzyme digestion. We found 28 (35%) cervical cancers contained ras mutations at H-ras codon 12, 49 (61%) at K-ras codon 12, and 5 (6%) at K-ras codon 13. There were no significant differences in incidence of the ras gene mutations among different histologic grades or clinical stages of the cancer (P > 0.05). This result suggests that ras mutation may be an important step involved in a substantial number of cervical carcinoma. The interaction of ras with other genes and/or events may also be involved in pathogenesis of this malignancy.

Loss of heterozygosity at the short arm of chromosome 3 in microdissected cervical intraepithelial neoplasia.

Loss of heterozygosity (LOH) is a common genetic finding in many human neoplasms, including cervical cancer. The detection of LOH at specific loci in the precursor of cervical cancer, cervical intraepithelial neoplasia (CIN) may help in elucidating the evolution of this cancer, which has a clearly defined histological premalignant phase. However, molecular genetic investigation of CIN is difficult because many of the lesions are very small and sometimes ill defined topographically. In this study we analyzed eighteen polymorphic microsatellite repeats on chromosome 3p in CINs using a method of primer extension pre-amplification (PEP) for whole genome amplification combined with microdissection. These markers encompass chromosome region 3pter-3p12. LOH at one or more loci was detected in five (33%) out of the 15 informative cases with low grade CIN (CIN 1), while 22 (92%) out of 24 cases with high grade CIN (CIN 2 and 3) (P<0.01). The highest incidence (41%) of LOH was detected at locus D3S1038 (3p26.1-3p25.2). Frequent LOH (more than 20%) was also found at other loci including D3S1110 (3p25.3-3p25.1) (31%), D3S656 (3p25.1) (24%), D3S1076 (3p21.2-3p21.1) (29%), D3S1300 (3p21.1-3p14.2) (24%), D3S1600 (3p14.2-3p14.1) (24%), and D3S1079 (3p13) (25%). The results from this study taken together with others indicate that the genetic alterations on chromosome 3p are common in high grade of CIN and are probably early events in cervical carcinogenesis. Tumor suppressor gene(s) that play a role in cervical neoplasm may be located on the short arm of chromosome 3, likely at or near 3p26.1-25.1, 3p21.2-21.1, and 3p14.2-13.

Methylation of p16INK4A in primary gynecologic malignancy.

The p16INK4A gene mapped on band p21 of chromosome 9 can be inactivated via multiple mechanisms including homozygous deletion, point mutation and promoter hypermethylation in various human tumors. A polymerase chain reaction (PCR) based analysis was performed to examine methylation of the p16INK4A gene promoter in 196 primary gynecologic malignancies including 98 cervical, 49 endometrial and 49 ovarian carcinomas. Methylation of p16INK4A was detected in 31% of cervical, 20% of endometrial, and 4% of ovarian carcinomas, respectively. The incidence of p16INK4A methylation in patients with cervical and endometrial carcinomas at advanced stages (stages III-IV) was statistically higher than those at early stages (stages I-II). There were also significant differences in the incidence of p16INK4A methylation in both cancers between the patients who had died of their disease or were alive with evidence of disease, and those without evidence of disease. The results indicate that methylation of the p16INK4A gene is present in a proportion of primary gynecologic malignancies and this alteration may be associated with poor outcome in cervical and endometrial carcinomas.

Alteration of cyclin D1 and CDK4 gene in carcinoma of uterine cervix.

Amplification and overexpression of cyclin D1 and CDK4 genes in cervical carcinoma were studied by semi-quantitative differential polymerase chain reaction assay and an immunostaining technique, respectively. Amplifications of cyclin D1 and CDK4 genes were found in 24% (27/113) and 26% (29/112) of tumors, respectively. Overexpression of cyclin D1 and CDK4 was demonstrated in 32% (21/66) and 73% (45/62) of tumors, respectively. No tumor showed CDK4 gene mutation on single strand conformational polymorphism. Sixteen percent (8/49) of the tumor specimens showed neither amplification nor overexpression. Disease stage, tumor grade and overexpression of cyclin D1 were found to be independent poor prognostic factors in cervical carcinoma.

Aberrant expression of p21(WAF1/CIP1) and p27(KIP1) in cervical carcinoma.

An immuno-histochemical study of p21 and p27 expression in cervical carcinoma was performed in 73 patients. Positive p21 and p27 staining was detected in 35.6 and 11% of tumour tissues, respectively. p21 expression was significantly correlated with advanced disease stage and negative human papilloma virus infection whilst positive p27 staining was not correlated with any clinical and pathological parameters studied. Kaplan-Meier estimation indicated that survival might be related to disease stage, tumour grade and p21 expression. Cox regression analysis confirmed that advanced stage disease and poorly differentiated tumour are independent prognostic factors for cervical carcinoma.

Protein profiling of cervical cancer by protein-biochips: proteomic scoring to discriminate cervical cancer from normal cervix.

Analysis of multiple proteins is thought to be essential for establishment of signature proteomic patterns that may distinguish cancer from non-cancer. Surface-enhanced laser desorption/ionization (SELDI) is an affinity-based mass spectrometric method in which proteins of interest are selectively absorbed to a chemically modified surface on a biochip. This technology may provide protein profiling of a variety of biological specimens. In this study, we explored whether the protein biochip SELDI approach could differentiate cervical cancer from non-cancer cohorts. We screened protein profiles generated by SELDI in 62 cervical epithelial cell samples microdissected from 35 invasive cervical cancer and 27 age-matched normal cervix tissue specimens, respectively. The cell lysates of pure populations of cervical cells were applied onto Ciphergen ProteinChip WCX2 Arrays. Proteins bound to the chips were analyzed on a ProteinChip Reader Model PBS II. Derived proteomic patterns were converted to a simple proteomic scoring for distinguishing cancer from non-cancer cohorts. SELDI protein profiles of cell lysates from 20 cervical cancer and 15 normal cervix tissue specimens were used to train and develop a classification scoring system that used a seven-protein mass pattern. The training samples could be correctly discriminated. When a test set of 27 samples was used for evaluation of this scoring system to distinguish cervical cancer from non-cancer, a sensitivity of 87%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 86% for the test population were obtained. All seven proteins appeared to be down regulated in cervical cancer. The results from this study indicate that the proteomics approach of SELDI mass spectrometry, in combination with a simple scoring system, may distinguish cervical cancer from its normal counterpart. If this approach is also workable in the analysis of cervical exfoliated cell lysate, it might potentially be used in the early diagnosis of invasive cervical cancer. In addition, the identification of these specific proteins in cervical cancer may also facilitate the discovery of new cervical tumor marker(s).

Clinical and pathologic significance of microsatellite instability in endometrial cancer.

Microsatellite instability (MSI) is identified as electrophoretic shifts in allele sizes of microsatellite DNA sequences. It is characteristic of a subset of sporadic colorectal tumors as well as hereditary nonpolyposis colorectal cancer (HNPCC). The cells that display MSI are thought to be susceptible to increased mutability. MSI has been detected in a wide variety of human tumors, but the influence of this form of genetic instability on disease initiation and progression remains unclear. Using a polymerase chain reaction (PCR)-based method we screened 50 sporadic primary endometrial carcinomas to characterize the prevalence of MSI in these tumors and analyze the correlation of MSI with clinicopathologic parameters in this malignancy. Fifteen cases (30%) displayed low frequency of MSI (MSI-L) showing MSI at one locus in 5 loci examined. Two cases (4%) showed high frequency of MSI (MSI-H) having MSI at 2 or more loci. Taking MSI-L and MSI-H cases together as MSI-positive, statistical analysis of patient age, tumor grade, and stage failed to disclose significant differences or trends between cases with MSI-positive and MSI-negative (P > 0.05). No significant relationship was observed between patients with MSI and without MSI (P > 0.05), however, the MSI exhibited only in those cases without evidence of disease at the 24th month after treatment. The difference is statistically significant when compared with patients who are alive with disease or died of disease (P < 0.01). However, the overall survival curves were not statistically different. We conclude that MSI is present in a subgroup of endometrial cancer.

Salivary oestradiol and progesterone after in vitro fertilization and embryo transfer using different luteal support regimens.

Salivary oestradiol (E2) and progesterone (P) levels have been shown to reflect the biologically active fractions in the serum. The luteal-phase status of stimulated cycles was investigated after in vitro fertilization and embryo transfer (IVF-ET). Thirty patients were randomly allocated to one of three luteal therapy groups: group A had no support, group B had intramuscular P and group C had intramuscular P and human chorionic gonadotrophin (hCG). One pregnancy was achieved in group A, two in group B and three in group C. Significant correlations between salivary and serum levels of E2 and of P in matched samples during luteal phase were found. Salivary E2 levels from luteal day 8 through day 14 and P levels from day 3 through day 14 were significantly higher in the pregnant than in the nonpregnant cycles. Among the nonpregnant cycles, salivary E2 and P levels were significantly higher in group C than in group A or B. These findings suggest that, in stimulated cycles for IVF-ET, determination of salivary E2 and P levels may be used as reliable alternatives to serum concentrations for assessing the luteal phase. Also, the additional hCG has an enhanced luteotrophic effect, as reflected by the higher salivary E2 and P levels, which may lead to a better pregnancy rate.

Salivary estradiol and progesterone during the normal ovulatory menstrual cycle in Chinese women.

Salivary and serum levels of estradiol and progesterone were measured by radioimmunoassay in 10 Chinese women during their normal menstrual cycles. Changes in salivary estradiol and progesterone levels followed a similar pattern to that in the serum. Significant correlation was found between salivary and serum levels of estradiol and progesterone (p less than 0.001). Measurements of these salivary steroids may be used to assess follicular dynamics. Moreover, salivary sampling is simple, convenient and stress free.

Microsatellite instability in cervical carcinoma.

OBJECTIVE: To investigate the incidence of microsatellite instability (MI) in cervical carcinoma and its relationship with clinicopathological characteristics. STUDY DESIGN: A retrospective study of 100 cases of cervical carcinoma. RESULTS: MI, defined as tumor-associated alterations in at least one of five dinucleotide microsatellite markers examined, was detected in 25% of the cervical carcinomas which were observed. There was a nonsignificant trend towards MI occurrence in higher grade tumors, more advanced stage and cases with poor clinical outcome. CONCLUSION: The results suggest that microsatellite instability is present in a subset of cervical carcinoma and may be an independent prognostic factor. Further research with more samples is required.

Expression of myeloperoxidase in acute myeloid leukemia blasts mirrors the distinct DNA methylation pattern involving the downregulation of DNA methyltransferase DNMT3B.

Myeloperoxidase (MPO) has been associated with both a myeloid lineage commitment and favorable prognosis in patients with acute myeloid leukemia (AML). DNA methyltransferase inhibitors (decitabine and zeburaline) induced MPO gene promoter demethylation and MPO gene transcription in AML cells with low MPO activity. Therefore, MPO gene transcription was directly and indirectly regulated by DNA methylation. A DNA methylation microarray subsequently revealed a distinct methylation pattern in 33 genes, including DNA methyltransferase 3 beta (DNMT3B), in CD34-positive cells obtained from AML patients with a high percentage of MPO-positive blasts. Based on the inverse relationship between the methylation status of DNMT3B and MPO, we found an inverse relationship between DNMT3B and MPO transcription levels in CD34-positive AML cells (P=0.0283). In addition, a distinct methylation pattern was observed in five genes related to myeloid differentiation or therapeutic sensitivity in CD34-positive cells from AML patients with a high percentage of MPO-positive blasts. Taken together, the results of the present study indicate that MPO may serve as an informative marker for identifying a distinct and crucial DNA methylation profile in CD34-positive AML cells.

Anti-inflammatory and analgesic effects and molecular mechanisms of JCICM-6, a purified extract derived from an anti-arthritic Chinese herbal formula.

The anti-inflammatory and anti-nociceptive effects and the molecular mechanisms of JCICM-6, a purified extract derived from an anti-arthritic Chinese herbal formula composed of Caulis Sinomenii, Aconiti laterralis Preparata, Rhizoma Curcumae longae, Radix Paeoniae albae, and Cortex Moutan, were examined for the first time. JCICM-6 was prepared using pharmaceutical extraction technology, purified by Amberlite XAD-7HP polymeric resin. Pharmacologically, in carrageenan-induced edema and carrageenan-evoked thermal hyperalgesia in paws of rats, the oral administration of JCICM-6 at dosages of 0.4, 0.8, and 1.6g/kg demonstrated significant inhibition with a dose-dependent manner. Mechanistic studies showed that JCICM-6 effectively decreased the production of the pro-inflammatory cytokines of IL-6 and IL-1beta and expression of COX-2 and iNOS proteins, and simultaneously elevated the level of anti-inflammatory cytokine IL-4 in the carrageenan-injected rat paw tissues and exudates. The positive reference drug, indomethacin at a dosage of 10mg/kg, demonstrated inhibitory potency in both rat models, but it could not augment the production of IL-4, indicating JCICM-6 and indomethacin might possess different pharmacological properties and molecular mechanisms although both have anti-inflammatory and analgesic effects in rats. These results suggest that JCICM-6 would be a valuable candidate for further investigation as a new anti-arthritic drug.